CALIBRACION DE LA SONDA FDR (Diviner 2000)

Salmonella is a rod-shaped bacterium that occurs ubiquitous in nature. Most strains in the soil are not dangerous to human or animal life. However, some of the Salmonellae are able to survive within their host and can cause disease in humans and animals. Salmonella infections are one of the most common food-related illnesses in the world and form a major problem for people in developing countries. In industrialized countries, the microorganisms are a threat especially to people with an impaired immune system. Symptoms of disease may vary from asymptomatic carriage in which the bacteria reside within the intestines and are shedded via the stool, to life-threatening sepsis in which the bacteria enter the blood stream. The type of disease is dependent upon the Salmonella strain the person gets infected with and the defense system of the host. Many infections are due to S. enterica serovar Enterica or serovar Typhimurium via food or water that is contaminated with animal waste.

Salmonella infection occurs through the ingestion of contaminated food or drinks. The acid in the stomach kills most of the ingested bacteria. However, in particular when the pH in the stomach is slightly higher than normal, some of the bacteria will pass the stomach. In the intestines, Salmonella has to compete with other defense mechanisms such as a thick mucus layer and the natural flora of the gut. If Salmonella succeeds in this task and survives, it eventually reaches the M cells in the Peyers’ patches of the small intestines. Salmonella invades these M cells and depending on the Salmonella strain, two types of disease can occur. During infection with the solely human typhoidal strains S. enterica serovar Typhi or serovar Paratyphi, the bacteria will spread to the underlying lymphoid tissues where they will infect macrophages. Next, the bacteria will enter the bloodstream and will reach the liver and spleen where Salmonella initiates a severe inflammatory response that is characteristic for typhoid fever and will be lethal in about 10-15% of the cases if not treated. However, when a person gets infected with the non-typhoidal strains such as S. enterica serovar Typhimurium, the bacteria usually stay within the Peyers’ patches and induce a local inflammatory response that is mediated by cytokines, chemokines, and neutrophils. This type of disease is called gastroenteritis and is characterized by diarrhea and is usually self-limiting. In certain groups of patients such as immunocompromised individuals, however, infection with this “harmless” bacterium may lead to severe disease. Another problem in this group of patients is the reactivation of

latent S. enterica serovar Typhimurium infection. Patients then suffer from recurring

infections with the same strain, as has been described for instance for AIDS patients. In many scientific studies on Salmonella an in vivo mouse model is used. Murine infection with S. enterica serovar typhimurium, causing gastroenteritis in humans, leads to disease in mice that is comparable to typhoid fever in man and therefore, serves a model for human infection with S. enterica Typhi or Paratyphi. The natural infection is the oral route, but experimental infections can also be induced by intravenous, intraperitoneal, or subcutaneous injection of bacteria. A soon as the bacteria have reached the blood, all these types of infections are similar and are very much alike that of the typhoid fever in

humans. The bacteria spread to the liver and spleen where they reside and multiply within macrophages. This, together with the influx of immune cells (macrophages and neutrophils) leads to enlargement of the liver and spleen (hepatosplenomegaly). Depending on Salmonella strain and dosage, an immune response is initiated that allows the mice to survive and that protects against secondary infection. Natural protection against S. enterica serovar Typhimurium infection is partly determined by the genes Ity and lps. Ity is the gene encoding Nramp1, a membrane phosphoglycoprotein that is expressed in macrophages of liver and spleen. Mice expressing the resistant allele (Nramp1G169) can control the initial in vivo replication of S. enterica serovar typhimurium allowing time for the development of a T cell-mediated immune response needed for clearance of the bacteria. However, mice expressing the sensitive allele Nramp1D169 _{cannot control the infection and}

will die. Expression of Nramp1 is enhanced upon activation of the macrophages by IFNγ

and LPS. Another gene protecting against Salmonella infection is lps, the gene encoding Toll-like receptor 4 (TLR4). TLR4 is a receptor involved in the recognition of lipopolysaccharide (LPS) that is abundantly expressed by Gram-negative bacteria such as Salmonella. Upon activation, an intracellular signaling cascade is initiated leading to the transcription of genes encoding cytokines such as IFNγ, Il-18 en TNFα. These cytokines play an important role in the activation of macrophages and thus in the defense against S. enterica serovar Typhimurium. Mice such as the C3H/HeJ strain that have a mutation in lps, cannot respond to LPS due to a non-functional TLR4 and as a consequence, are extremely sensitive to S. enterica serovar Typhimurium infections.

In defense against S. enterica serovar Typhimurium macrophages play a major role. Macrophages are phagocytic cells that contain a multitude of antimicrobial defense mechanisms. However, S. enterica serovar Typhimurium has evolved to survive and even replicate within this hostile environment. An important defense mechanism of macrophages against microorganisms is the production of oxygen radicals. These radicals are highly toxic and would probably result in bacterial death if Salmonella had not adapted. In the genome of Salmonella there are two Salmonella pathogenicity islands (SPI-1 and SPI-2) that encode type III secretion systems (TTSS). The first TTSS encoded by SPI-1 is involved in the uptake by the host cell and encodes a kind of syringe apparatus through which certain proteins can be injected into the host cell cytosol thereby inducing changes in the cell membrane that leads to the uptake of the bacterium, even by non-professional

phagocytes. This process is called Salmonella-induced uptake. Salmonella enters a

vacuole where it can survive due to the induction of the second TTSS encoded by SPI-2. In this process proteins are injected into the host cell cytosol and lead to the disturbance of essential antibacterial processes, allowing Salmonella to survive. For instance, SPI-2 encoded proteins prevent the translocation and assembly of the active NADPH oxidase complex and in this way, Salmonella can prevent the production of superoxide. Besides SPI-1 and SPI-2 encoded proteins, other genes such as soxR/S and phoP/Q play an important role in defense against superoxide and in survival within the host.

Chapter 1 gives an overview of what is currently known about Salmonella, the interaction with the host and systems that play a role in the defense against superoxide and in survival within macrophages.

In Chapter 2 the development is described of an in vivo model for reactivating S.

enterica serovar Typhimurium infection through total body irradiation or CD4+ _{T cell}

depletion. An important complication of S. enterica serovar Typhimurium infection in certain groups of patients is the recurrence of infection. The infection is cleared, but Salmonella may reside within the body despite the host immune system, and will strike again as soon as the immune system is impaired. It is unknown at which niche Salmonella hides and which processes play role in the suppression of growth during the phase of persistency. Reactivating Salmonella infections have been mainly described for patients who underwent irradiation or received glucocorticoids and for individuals with AIDS or other immune impairments. Our model shows that CD4+ _{T cells play a role in the suppression of growth}

of S. enterica serovar Typhimurium during the phase of persistency.

Since a couple of years, patients suffering from rheumatoid arthritis or Crohn’s disease are being treated with the tumor necrosis factor α (TNFα) neutralizing antibodies Infliximab or Ethanercept. This type of treatment has proven to be of great benefit to these patients, but makes them susceptible to primary as well as reactivating infection with intracellular bacteria such as Mycobacterium tuberculosis. TNFα is a cytokine that plays an important role in the activation of macrophages and in defense against pathogens including

Salmonella. For Salmonella infections it is known that neutralization of TNFα leads to

increased risk of severe infections, but whether such treatment may also lead to reactivation of a latent Salmonella infection, as in latent Mycobacterium tuberculosis

infection, is not clear. In Chapter 3 we investigated whether TNFα plays a role in the

suppression of S. enterica serovar Typhimurium during the phase of persistency in mice. In the model used, we did not observe reactivation of the S. enterica serovar Typhimurium infection after treatment with antibodies to TNFα. In addition, we observed that addition of

anti-TNFα to IFNγ-stimulated mouse macrophages (RAW264.7) had no effect on the IFNγ

induced effect of reduced outgrowth of S. enterica serovar typhimurium. This could mean that TNFα plays a minor role compared to IFNγ during infection with S. enterica serovar

Typhimurium. As long as IFNγ produced by CD4+ _{T cells remains present, latent S.}

enterica serovar Typhimurium infection will be suppressed and reactivation will not occur. In Chapter 4 we describe the research on bacterial mutants that are able to survive for a longer period of time within macrophages. We have created S. enterica serovar Typhimurium mutants by random P22 MudJ transposon insertion. In this way we have created several mutants that were selected for the ability to survive for a longer period of time than wild-type S. enterica serovar Typhimurium within mouse macrophages. Eventually, two mutants were selected that after inverse PCR and sequence analysis appeared to be the same. The MudJ transposon had inserted into rmlC, the gene encoding dTDP-4-deoxyrhamnose 3,5-epimerase, an enzyme involved in the formation of the O- antigen of the lipopolysaccharide (LPS). Analysis of the LPS showed that this mutant had truncated LPS and was very similar to an S. enterica serovar Typhimurium strain of the

rough Ra chemotype. This is a mutant lacking the O-antigen and contains only the lipid A and the core region of the LPS. Also the Ra mutant was able to survive for a longer period of time in RAW264.7 macrophages; even after 48 h high numbers of bacteria could still be detected. Despite this ability to survive longer, these mutants were not capable of inducing severe infection in mice. These LPS mutants are killed in vitro by rat or human complement, and likely, the attenuated phenotype in mice can be explained by this increased in vitro susceptibility to complement, although we could not confirm this with high numbers of bacteria in mouse serum that has low levels of complement.

By random MudJ transposon insertion in wild-type S. enterica serovar Typhimurium 14028s we have created mutants that were next selected for their susceptibility to intracellular superoxide, as described in Appendix 1. By inverse PCR and sequence analysis we determined the position in the genome where the MudJ transposon had been inserted and which gene might have been inactivated. One of the mutants obtained in this way was studied in further detail and has been described in Chapter 5. In this mutant, designated AVD101, the MudJ transposon had inserted into the promotor region of pnp, the gene encoding PNPase. This protein is involved in the degradation of mRNA and in the growth adaptation at low temperatures and it is considered a regulator of virulence and persistence of S. enterica serovar Typhimurium. We described an additional role for PNPase in the resistance to superoxide and for intracellular survival within macrophages.

In the research on superoxide-resistance genes we describe in Chapter 6 the isolation and characterization of DLG294, an S. enterica serovar Typhimurium mutant in which, through MudJ transposon insertion, an as yet unknown gene was inactivated. This gene was designated sspJ. The protein SspJ is no longer produced by this mutant resulting in increased susceptibility of this mutant to menadione, a redox cycling agent that generates superoxide radicals within the bacterium. DLG294 appeared to be attenuated in vitro in macrophages and in vivo in mice. By constitutive expression of sspJ on a plasmid the phenotype of DLG294 was restored to that of the wild-type strain. This confirmed the role of SspJ in the defense against superoxide and in virulence. However, the exact role and functioning of SspJ is not clear yet.

DLG294 was next studied in vivo in mice, as described in Chapter 7. DLG294 induced hardly any granulomatous lesions in the liver after subcutaneous infection of Salmonella- resistant (Ityr) C3H/HeN mice with 3 × 104 CFU and the numbers of bacteria were 3 log units lower than those of the wild-type strain on day 5 after infection. However, DLG294 appeared to be as virulent as the wild-type strain and induced similar liver pathology in

p47phox-/- _{mice. These mice lack a functional NADPH oxidase system because of a lack of}

p47phox and as a result cannot produce superoxide. Also in bonemarrow-derived

macrophages of these p47phox-/- _{mice and in X-CGD PLB985 cells the bacterial numbers of}

DLG294 were as high as those of the wild-type strain. These results suggest that SspJ plays a role in resistance against oxidative stress and in survival and replication of S. enterica serovar Typhimurium both in vitro and in vivo.

Macrophages play an important role in Salmonella infections. They exert a dual role; that of a host cell possibly hiding the bacterium from the hostile exterior, and that of an

effector cell in acquired immunity. DLG294 is more sensitive to superoxide and attenuated in macrophages, and we hypothesized that hypersusceptibility to superoxide plays a causative role in its attenuated behavior. However, other processes might play a role as well leading to the reduced ability of DLG294 to survive within macrophages. Infection with S. enterica serovar Typhimurium leads to the activation of the macrophages to kill and eliminate the bacteria. Diverse mechanisms play a role in this activation process. In Chapter 8 we have investigated the effect of wild-type and DLG294 infection on the gene expression in macrophages using Affymetrix gene chips. Using these chips, the expression of 6,400 genes can be studied simultaneously. Wild-type S. enterica serovar Typhimurium and DLG294 appeared to influence the expression of many genes, however, no differences between the two types of infected cells were apparent. From this we concluded that the reduced outgrowth of DLG294 in macrophages must be attributed to the mutation in sspJ and not to a different, indirectly induced, activation status of the macrophages compared to that of wildtype-infected macrophages.

To further characterize DLG294, we have studied the in vitro phenotype of DLG294 further in Chapter 9 using a phenotypical array in which several hundreds of processes can be studied at the same time. This makes it possible to compare the in vitro phenotype of DLG294 to that of the wild-type strain. Also, we have looked at the intracellular gene expression profile of DLG294 in RAW264.7 macrophages and compared that to intracellular wild-type S. enterica serovar Typhimurium. The phenotypical array revealed that DLG294 gained the ability to use nitrogen sources for growth, has hampered resistance to several antibiotics, and shows increased susceptibility to acidic and alkalic pH. Comparison of the gene expression profile of intracellular DLG294 with that of the wild- type strain revealed only a few differences. Most likely, DLG294 has reduced membrane integrity that leads to increased uptake of toxic compounds and as a result, more damage to the bacterium.